DNA encoding mannose 6-phosphate reductase and recombinants produced therefrom

ABSTRACT

DNA encoding mannose 6-phosphate reductase (M6PR) and the use of the DNA in vectors and bacteria and in plants. The enzyme enables the production of mannitol in plants which increases stress tolerance, particularly to salt.

GOVERNMENT RIGHTS

This invention was developed under U.S.D.A. Contract No. 93-37100-8907and 94-01439. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a DNA encoding mannose 6-phosphatereductase (M6PR) which is part of the pathway which forms mannitol inplants. The DNA encoding M6PR was isolated from celery. When the DNA istransformed into a plant the resulting transformed plant can be moretolerant to environmental stresses in the form of dehydration, salinityand drought.

(2) Description of Related Art

In many plants, sucrose and starch are the primary products ofphotosynthetic carbon assimilation. In other species, however, acrylicpolyols (e.g., sorbitol, mannitol) can also be primary products andaccount for between 15 and 60% of the assimilated carbon, depending onthe species (Loescher, W. H., Physiol. Plantarum 70:553-557 (1987); Fox,T. C., et al Plant Physiology 82:307-311 (1986); Flora and Madore Planta189:484-490 (1993); Loescher, W. H. and Everard, J. D., PhotoassimilateDistribution in Plants and Crops, 185-207 (1996)), the stage of leafdevelopment, (Davis, J. M., et al., Plant Physiol, 86:129-133 (1988) andenvironmental factors (e.g., salinity (Everard, J. D., et al., PlantPhysiol, 106:281-292 (1994)) and water stress; Escobar-Gutierrez, A., etal., Plant Physiol suppl 105: abstract 575 (1994)). The influence ofdevelopmental and environmental factors suggest that the partitioning ofphotoassimilates between sugar alcohols, sucrose and starch is understrict metabolic control. This is consistent with the complexity anddiversity of the control mechanisms known to govern sucrose and starchsynthesis in species that do not synthesize sugar alcohols (Quick, W. P.and Schaffer, A. A., Sucrose metabolism in sources and sinks. In:Photoassimilate distribution in plants and crops: source-sinkrelationships. Zamski, E., and Schaffer A., (eds), Marcel Dekker, Inc.:pp 115-156 (1996); and Preiss, J. and Sivak, M. N., Starch synthesis insinks and sources. In: Photoassimilate distribution in plants and crops:source-sink relationships. Zamski, E., and Schaffer A., (eds), MarcelDekker, Inc.: pp 63-96 (1996)). There is, however, almost no equivalentinformation on the mechanisms by which polyol metabolism is regulated orintegrated with these other pathways in sugar alcohol synthesizingspecies. Such mechanisms are of more than just esoteric interest since:(a) an estimated 30% of global annual carbon assimilation results inpolyol production (Bieleski, R. L., Sugar alcohols. In: F. A. Loewus andW. Tanner, eds., Plant Carbohydrates I. Intracellular Carbohydrates,Encyc. Plant Physiol. Vol. 13A, New Series, Springer-Verlag, NY, pp.158-192 (1982)), (b) many polyol producing species are of considerableeconomic value, and (c) substantial correlative evidence shows thatpolyols accumulate in higher plants subjected to stresses mediated atthe cellular level by changes in water activity, suggesting a role instress tolerance (Bohnert, Hans J., et al., The Plant Cell, 7:1099-1111(1995); Ahmad, I., et al., New Phytol 82:671-679 (1979); Hirai, M.,Plant Cell Physiol. 24:925-931 (1983); Everard, J. D., et al., PlantPhysiol 106:281-292 (1994); Stoop, J. M. H., et al., Plant Physiol.106:503-511 (1994)).

In recent years major advances in the understanding of sugar alcoholmetabolism in higher plants have been facilitated by the detection,characterization, and purification of several key enzymes (see Loescherand Everard, Photoassimilate Distribution in Plants and Crops, 185-207(1996) for a review). The successful cloning of genes for NADP-dependentsorbitol 6-phosphate reductase (Kanayama, Y., et al., Plant Physiology100:1607-1608 (1992)), mannose 1-oxidoreductase (Williamson, J. D., etal., Proc. Natl. Acad. Sci. 92:7148-7152 (1995)) and the introduction ofheterologous genes, which confer sugar alcohol synthesis to plants thatnormally do not produce them (Tarczynski, M. C., et al., Science259:508-510 (1993); Thomas, J. C., et al., Plant Cell and Environ18:801-806 (1995); Tao, R., et al., Plant Cell Physiol 36:525-532(1995)), now provide powerful tools with which to study sugar alcoholbiochemistry and physiology. For example, Tarczynski, M. C., et al.(Science 259:508-510 (1993)), and Thomas, J. C., et al. (Plant Cell andEnviron 18:801-806 (1995)) have recently shown that transgenic tobaccoand Arabidopsis plants expressing the bacterial mannitol dehydrogenase(mtlD) gene not only produce low levels of mannitol, but also exhibitenhanced sodium chloride tolerance. In another study the inhibitoryeffects of 300 mM NaCl on the growth of celery suspension cultures weresubstantially reduced when mannitol, rather than sucrose, was includedas the sole carbon source (Pharr, D. M., et al., Plant Physiology180-194 (1995)). Such studies convincingly demonstrate that polyolsconfer some stress protection but give little insight into theunderlying mechanisms. Mechanisms are beginning to be elucidatedhowever, mannitol accumulation in salt stressed celery plants has beenassociated with a down regulation (at both the mRNA and protein levels)of mannitol dehydrogenase (MTD), a key catabolic enzyme (Williamson, J.D., et al., Proc. Natl. Acad. Sci., 92:7148-7152 (1995)), although,enhanced de novo synthesis is also undoubtedly involved in thisacclimation response (Everard, J. D., Plant Physiol. 106:281-292 (1994);Loescher, W. H., et al., Plant Physiology, 170-178 (1995)). Otherevidence showing a possible role for polyols in stress metabolism inhigher plants includes the identification of an aldose reductase inCraterostigma leaves and barley embryos that accumulates when thesetissues undergo desiccation (Bartels, D., et al., EMBO J 10:1037-1043(1991)).

A pathway for mannitol synthesis in higher plants has been establishedin celery (Rumpho, M. E., et al., Plant Physiol. 73:869-873 (1983)) andappears to be present in other mannitol synthesizing species (Harloff,H. J., et al., J. Plant Physiol 141:513-520 (1993)). Biosynthesisinvolves three unique enzymatic steps consisting of an isomerization(F6P to mannose 6-P, mediated by mannose 6-P isomerase), a reduction(mannose 6-P to mannitol 1-P by mannose 6-P reductase (M6PR)) and adephosphorylation (mannitol 1-P to mannitol, by mannitol 1-Pphosphatase). Radiotracer studies and kinetic analyses indicate thatM6PR plays a regulatory role in this pathway. This enzyme has beenpurified and partially characterized (Loescher, W. H., et al., PlantPhysiol. 98:1396-1402 (1992)).

U.S. Pat. No. 5,268,288 to Pharr describes mannitol oxidoreductaseprotein. The enzyme converts mannitol to mannose in plants. It is thusdifferent from the present invention which relates to mannitolproduction. The patent describes various recombinant techniques usefulin the present invention. U.S. Pat. No. 5,492,820 to Sonnewald et aldescribes plasmids (vectors) for producing recombinant plants withaltered sugar expression. The disclosure of such vectors is incorporatedinto the disclosure of the present application as well.

OBJECTS

It is therefore an object of the present invention to provide DNAencoding mannose 6-phosphate reductase (M6PR) which is useful inproducing plasmids and transgenic plants with increased tolerance toenvironmental stresses, particularly salinity. These and other objectswill become increasingly apparent by reference to the followingdescription and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of an electrophoresis gel showing in vitrotranslation of poly(A)+RNA isolated from celery leaf tissue. Lanes 1 and2 are Coomassie Brilliant blue R250 stained lanes from a 12.5%polyacrylamide gel showing Bio-Rad (Richmond, Calif.) molecular weightmarkers and purified celery leaf M6PR (0.1 μg), respectively. Lanes 3and 4 show an autoradiograph of a subsample of the in vitro translationproducts represented from leaves 5 and 7 after immunoprecipitation withM6PR-specific antisera; all lanes were from the same gel.

FIGS. 2A, 2B and 2C show verification of M6PR specific clones. FIG. 2Ais a graph showing M6PR activities in sonicated extracts of onenonspecific and three putative M6PR clones with and without IPTGinduction. FIG. 2B is a photograph of an SDS gel of the extracts (100μg/lane); the extreme right-hand lane contains 3 μg of authentic celeryleaf M6PR. FIG. 2C is a photograph of a Western blot; an SDS gelcontaining 5 μg total protein per lane was blotted to PVDF membrane andprobed with M6PR-specific antisera, as in Panel C the extreme left-handlane contains authentic leaf enzyme.

FIGS. 3A and 3B are drawings showing the base and translated amino acidsequence of celery cDNA clone D. The open reading frame (M6PR ORF) codedfor a peptide of 35.2 kD and had three domains which were identified,through computer data base searches, as being typical of the aldoketoreductase family. Also shown is the peptide resulting from trypticdigestion of authentic celery leaf M6PR and the Xho 1 restriction sitewithin the coding region. Two other independent clones were sequenced onboth strands and only differed from the displayed sequence in thelengths of their 3′ and 5′ non-coding regions.

FIGS. 4A and 4B are drawings showing an amino acid sequence comparisonof M6PR and NADP-sorbitol-6-phosphate dehydrogenase from apple (NCBIaccession # D11080). Sequences were 64% identical (shown by enclosedareas) and showed 84% similarity, if the functional relatedness of theresidues was considered. For the latter comparison the following groupswere used: acidic (D,E), basic (H,K,R), hydrophobic (A,F,I,L,M,P,V,W)and polar (C,G,N,Q,S,T,Y); “.” indicates lack of functional similarity.

FIG. 5A is a photograph of SDSPAGE gel (12.5% acrylamide) of samplescollected after the various purification steps (See Table 2). Lanes: 1and 7, Bio-Rad molecular mass markers; 2, 27,500 g supernatant ofdisrupted cells (80 μg protein); 3, 30 to 60% acetone fraction (120 μg);4, post gel-filtration chromatography on Sephacryl S-200 (30 μg); 5,post affinity chromatography on Reactive Yellow 86 eluted with 0.1 mMNADPH (15 μg); 6, product of a second pass over the RY86 column elutedwith a linear gradient (0 to 0.2 mM) of NADPH (2 μg). 8, authenticcelery leaf M6PR (0.5 μg). FIG. 5B is a photograph of a Western blot ofproteins from selected purification steps (see above), probed withM6PR-specific antisera. Lanes: 1, 6 and 7 authentic celery leaf M6PR; 2,crude supernatant; 3, post S200 gel filtration; 4 and 5, after 1 and 2passes over RY86 affinity column, respectively.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a DNA encoding mannose-6-phosphatereductase (M6PR) free of any other DNA as set forth in SEQ ID NO:1.

The present invention also relates to a DNA encoding mannose-6-phosphatereductase (M6PR) in a plasmid in an Escherichia coli as contained in adeposit identified as ATCC 98041.

The present invention also relates to a recombinant plasmid containingDNA encoding mannose-6-phosphate reductase as set forth in SEQ ID NO:1.

The present invention also relates to a recombinant plasmid containingDNA encoding mannose-6-phosphate reductase in a plasmid in E. coli ascontained in a deposit identified as ATCC 98041.

The present invention also relates to a bacterium containing arecombinant plasmid containing DNA encoding mannose-6-phosphate as setforth in SEQ ID NO:1.

The present invention also relates to a bacterium containing arecombinant plasmid containing DNA encoding mannose 6-phosphate in theplasmid in an E. coli as contained in a deposit identified as ATCC98041.

The present invention also relates to a method for detecting unknown DNAencoding mannose-6-phosphate reductase (M6PR) which comprises probingthe unknown DNA with a probe DNA encoding a unique region of the M6PR asset forth in SEQ ID NO:1.

The present invention also relates to a method for detecting an unknownDNA encoding mannose-6-phosphate reductase (M6PR) which comprisesprobing the unknown DNA with a probe DNA encoding a unique region of theM6PR in a plasmid in E. coli as contained in a deposit identified asATCC 98041.

The present invention also relates to a transgenic plant containingrecombinant DNA encoding mannose-6-phosphate reductase (M6PR) based uponSEQ ID NO:1. The antisense of SEQ ID NO:1 is used.

The present invention also relates to a transgenic plant containingrecombinant DNA encoding mannose-6-phosphate reductase (M6PR) in aplasmid in an E. coli contained in a deposit identified as ATCC 98041.

The present invention also relates to a method for detecting mannose 6-Preductase (M6PR) which comprises:

(a) reacting an antibody selective for binding to the M6PR for screeningan expression library suspected of encoding the M6PR; and

(b) detecting the antibody binding to the M6PR.

The present invention also relates to a method for producing mannose6-phosphate (M6PR) which comprises:

(a) providing a bacterium containing a recombinant DNA encodingmannose-6-phosphate reductase (M6PR) free of any other DNA as set forthin SEQ ID NO:1 in a culture medium;

(b) expressing the M6PR in the culture medium; and

(c) isolating the M6PR.

The present invention also relates to a method for producing mannose6-phosphate reductase (M6PR) which comprises:

(a) providing a bacterium containing a recombinant plasmid containingDNA encoding mannose 6-phosphate reductase in the plasmid in an E. colias contained in a deposit identified as ATCC 98041 in a culture medium;

(b) expressing the M6PR in the culture medium; and

(c) isolating the M6PR.

The cloning of cDNAs encoding M6PR and the partial purification andcharacterization of active M6PR isolated from transformed E. coli isdescribed.

The DNA of celery (Apium graveolens) encoding M6PR was deposited withthe American Type Culture Collection on Apr. 30, 1996 in the BLUESCRIPTUNI-ZAP XR (Stratagene, Lajolla, Calif.) plasmid described and claimedin U.S. Pat. No. 5,128,256 contained in Escherichia coli SOLR anddeposited as ATCC 98041. The plasmid contains a 1.3 kbp insert which iscleaved with Eco R1 and Kpn1 restriction enzymes. The culture is grownin the presence of 50μ/ml ampicillin in LB media. No rights are grantedto the DNA encoding M6PR except those accorded by the Budapest Treaty.

For M6PR, DNA encoding sequence is shown in SEQ ID NO:1. The encodedM6PR is shown in SEQ ID NO:2 and in FIGS. 3A and 3B. For aldose6-phosphate reductase (S6PR), the DNA sequence is shown in SEQ ID NO:3and the encoded S6PR is shown in SEQ ID NO:4. FIGS. 4A and 4B show thealignment of M6PR and S6PR as set forth in SEQ ID NO:2 and SEQ ID NO:4.

EXAMPLE 1

Isolation of M6PR Encoding DNA

Methods

RNA Isolation and Poly(A)+RNA Selection

Total RNA was extracted from approximately 10 g samples of the fifth andseventh leaves of mature celery (Apium graveolens c.v., Giant Pascal)plants, according to Gilmour, S. J., et al., Plant Physiol. 87:745-750(1988)). Slight modifications includes: (a), addition of the polyphenoloxidase inhibitors cupferron (1 mM) and 2-mercaptobenzothiazole (1μg/ml) to the extraction buffer immediately to prior use; (b), inclusionof three phenol:chloroform:isoamyl alcohol (25:24:1 v:v:v) partitioningsteps on the aqueous phase, followed by three chloroform/isoamyl alcohol(49:1 v:v) partitionings to remove residual phenol; (c), a single LiClprecipitation followed by four ethanol precipitation steps. Total RNAyields (as determined by OD₂₆₀) averaged 500±180 μg/gFwt. The absence ofcontaminating DNA was confirmed on an agarose gel after RNAasetreatment.

Poly (A)+RNA was isolated by oligo-dT-cellulose chromatographyessentially as described by Sambrook et al (Molecular cloning. ColdSpring Harbor Laboratory Press, NY (1989)) but using a modified loadingbuffer (0.12 M NaCl, 0.01 M Tris-HCl pH 7.5, 0.001 M EDTA). Poly(A)+RNAwas eluted from the column in the above buffer with the NaCl omitted.

In vitro translation and immunoprecipitation

1 μg of poly(A)+RNA from each leaf was translated in vitro using rabbitreticulocyte lysates (Promega Corp., Madison, Wis.) and [³⁵S]methionine, according to the suppliers directions. Translation products,after 1 h at 30° C., were separated by SDS-PAGE (Laemmli, U. K., Nature227:401-407 (1970)) either, before (total) or after, immunoprecipitation(Hondred, D., et al., Plant Mol. Biol., 9:259-275 (1987)) withM6PR-specific antisera (Ried, J. L., et al., BioTechniques 12:660-666(1992)).

cDNA Library Construction

A unidirectional cDNA expression library was constructed in UniZap™ XRvector (Stratagene, LaJolla, Calif.) using a mixture of poly(A)+RNA fromleaves 5 and 7 (2.5 μg of each). After packaging the phage library wasamplified once before screening.

Library Screening

Two hundred thousand plaque forming units (pfu's) were screened for M6PRexpression at a density of 40,000 pfu's per 140 mm Petri dish. Oncephage plaques became visible (after approximately 3 h at 42° C.),nitrocellulose disks (previously soaked in 10 mM IPTG and thenair-dried) were laid onto the surface of the plates and the cultureswere incubated for a further 3.5 h at 37° C. The membranes were replacedwith fresh IPTG soaked membranes and the cultures were incubated for afurther 3 h. Both sets of membranes were screened using M6PR-specificantisera (Ried et al., BioTechniques 12:660-666 (1992)) at a dilution of1:10,000 (see Everard et al., Plant Physiol 102:345-356 (1993), formethods used). Over 100 plaque giving positive signals in the initialscreening were recovered from the plates and ten of these were subjectedto two additional rounds of screening. Twelve other recombinant (asdetermined by α-complementation; Sambrook et al., Molecular cloning.Cold Spring Harbor Laboratory Press, NY (1989)) plaques that did notgive a positive reaction with M6PR antisera were also selected asnon-specific control clones.

After selection through three rounds of screening, clones (bothputative-M6PR and nonspecific) were in vivo excised to yield phagemid(plasmid) clones in E. coli strain SOLR (Stratagene, La Jolla, Calif.).It should be pointed out that although 10 individual M6PR-putativeclones were selected for further study it is not certain that theserepresented 10 individual mRNA's isolated from the original populationin the leaf material used. This is because the original library wasamplified once before screening (see above).

Sequencing of M6PR clones

Three putative-M6PR clones were sequenced on both strands with anApplied Biosystems 373A sequencer using dye-primer and dye-terminatormethodologies. Sequences were obtained using T3, T7 and 20-mer primerscorresponding to internal sequences. Consensus sequence was derived bymatching the two strands of each individual clone and by comparison ofthe three independent clones.

Sequence analysis was performed using SeqEd (Applied Biosystems Inc.,Foster City, Calif.) and Lasergene (DNASTAR Inc., Madison, Wis.).Sequence comparisons with other databases was performed through theNational Center for Biotechnology Information via the BLAST server.Peptide comparisons were made through EXPASY-Prosite and PRINTS.

Clone Confirmation

Internal Peptide Sequencing

M6PR was found to be unsuitable for amino acid sequencing in the nativestate, presumably because of an N-terminus block. M6PR purified asdescribed by Loescher et al., Plant Physiol. 98:1396-1402 (1992) wasfurther purified by running approximately 200 mg on a preparative 10polyacrylamide gel under denaturing conditions. After staining withCoomassie Blue R250 (0.05%, wt:v in acetic acid:methanol:H₂O; 10:40:50,v:v:v) for 2 minutes and destaining (in stain solvent alone) the band ofgel containing the M6PR was excised and electroeluted (Hunkerpillar etal., Methods in Enzymology 91:227-236 (1983)). The eluted protein wasdried in vacuo and taken up in 80% ethanol (to remove residual SDS). Theprecipitated protein was pelleted by centrifugation, and the pelletdissolved in 100 mM ammonium bicarbonate (pH 8.2) prior to digestionwith trypsin at 37° C. for 16 h. Trypsin was added in two equal doses(2%, by weight at each addition), with the second dose added after 8 hdigestion.

Digestion products were separated by reverse phase chromatography on a1×25 mm column (Applied Biosystems) eluted in a 90 minute lineargradient of TFA (0.1% v:v in H₂O) and acetonitrile (90:9.91/5:0.085;acetonitrile: H₂O: TFA v:v:v), at a flow rate of 830 nl/sec. Prominentpeptides (as detected by OD_(212nm)) were collected, dried down invacuo, and subjected to amino acid sequencing on an Applied Biosystems477A sequencer.

Test for M6PR Activity in Putative Clones

Three putative M6PR and one non-positive clone (as determined byantibody screening) were tested for M6PR activity. Duplicate 10 mlcultures of each clone were grown in LB+ampicillin (amp; 50 mg/ml). Oncean average OD₆₀₀ of 0.5 had been attained one culture of each pair wasinduced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG;final concentration 10 mM). Control (uninduced) cultures had an equalvolume of sterile water added. Cultures were maintained at 30° C. for afurther 3 h and were harvested at an average OD₆₀₀ of 1.29±0.02. Cellswere pelleted by centrifugation (2000×g for 5 min) and washed twice byresuspending the pellets in 5 ml Tris (100 mM, pH 7.5) containing 250 mMPMSF, with a centrifugation step between each wash. After the final washpelleted cells were maintained on ice. Just prior to disruption thecells were resuspended in 4 ml of extraction buffer (100 mM Tris-HCl pH7.5, 250 mM PMSF, 10 mM DTT and 0.1 Triton X-100) and transferred toblood dilution vials (American Scientific Products, McGaw Park, Ill.).Cells were ruptured by suspending the vial containing the cells 0.5 cmabove the cuphorn probe of a Heat Systems W-385 A5 sonicator (MisonicsFarmingdale, N.Y.) and subjecting them to 2 minutes sonication at fullpower. Coolant was circulated around the vial to maintain thetemperature at approximately 4° C. After sonication, 1.5 ml aliquots ofthe homogenate were centrifuged at 13,000×g for 5 minutes at 4° C.Supernatants were assayed for M6PR activity (Loescher et al., PlantPhysiol. 98:1396-1402 (1992)); with at least two different aliquotvolumes from each extract to test for linearity. Supernatant aliquots(containing approximately 100 mg of total protein) were precipitated byadding a 7 times volume of acetone. After standing overnight at −21° C.the samples were prepared by SDSPAGE and Western blotting as describedin Everard et al., Plant Physiol 102:345-356 (1993).

Large Scale PreTarations and Purification

One liter LB/amp cultures were initiated by the addition of 50 ml of anovernight culture of clone D (initial OD₆₀₀, approx., 0.15). At an OD₆₀₀of approximately 0.25, IPTG was added to a final concentration of 1 mMand the culture was incubated by 30° C. until OD₆₀₀ was between 0.8 and1.0 (after approximately 6 hours; a preliminary experiment showing thatM6PR specific activity began to level off when OD₆₀₀>1.0). Cells wereharvested by centrifugation and, after washing as described above, werefrozen at −80° C. Prior to extraction, cells were thawed slowly on iceand ruptured by either: 1. Sonication: cells were suspended in 20 mlextraction buffer (see previous section for composition) in a blooddilution vial and sonicated for 5 min at full power. The homogenate wascentrifuged (20,000×g for 10 min), the supernatant collected, and thepellet resuspended in 10 ml extraction buffer and sonicated for afurther 5 min, this cycle was repeated three times: or 2.Decompression/shearing: Cells suspended in 20 ml extraction buffer wereruptured by three passes through a French Press followed bycentrifugation (20,000×g for 20 min).

Subsequent purification steps were essentially as described in Loescheret al., Plant Physiol. 98:1396-1402 (1992) except for Reactive Yellow 86(RY86) chromatography. Here the active fraction from the gel gofiltration step was split in two, and each fraction run separately onthe RY86 column as described in Loescher et al., Plant Physiol.98:1396-1402 (1992). The active fractions were pooled and either dilutedby the addition of 0.5 v of column buffer or desalted using centrifugalconcentrating devices (30 kD cut-off membranes) and washed with 2volumes of column buffer. This step was performed to dilute or removeNADPH. The sample was then loaded back onto the RY86 column and activityeluted in a linear gradient between 0 and 0.2 mM NADPH in column buffer.The purified M6PR was desalted and concentrated using centrifugalfiltration and was either used immediately for kinetic characterizationor stored at −210° C. after adding glycerol (1:1, v:v).

Protein Determinations

Protein content was determined by the method of Bradford, M. M., AnalBiochem 136:248-254 (1976) using Bovine Serum Albumen (BSA) as astandard.

Results

Characteristics of M6PR-Specific Immnunoprecipitationi Products

FIG. 1 shows that immunoprecipitation (with M6PR-specific antisera) ofthe in vitro translation products synthesized from poly(A)+RNA isolatedfrom leaves 5 and 7 yielded a single dominant peptide (molecular mass,35.1 kD) from each leaf. The molecular mass of authentic celery M6PR runon the same gel (but not immunoprecipitated) was 35.1 kD;immunoprecipitation prior to SDSPAGE had no effect on the relativemobility of M6PR (data not shown). Five and seven percent of the totalTCA precipitatable radioactivity was recovered in theimmunoprecipitation products from leaves 5 and 7, respectively.

Characteristics of the cDNA Library

The primary library consisted of >1.5×10⁶ plaque forming units (pfu's)of which 0.33w were non-recombinant, as estimated by α-complementation.Phagemids from 12 randomly selected recombinant clones were in vivoexcised and used to transform E. coli (strain SOLR). The average insertsize (after digestion with Eco R1 and Xho 1) was 1.7 kb, with a sizerange between 1.0-2.3 kb.

Library Screening

Of the 200,000 pfu's screened an estimated 0.15+0.04% gave a positivesignal with M6PR-specific antisera and were thus identified as putativeM6PR clones. Ten of these were subjected to two more rounds of screeningand three of these were characterized and their authenticity as M6PRclones was confirmed as described below.

Expression of M6PR Enzyme Activity

FIG. 2A shows the activity of M6PR, in one non-specific (2) and thethree putative M6PR clones (B, C and D), with and without induction withIPTG. Under IPTG induction, the putative M6PR-specific clone expressedM6PR activity, whereas little or no activity was observed in thenon-specific clone (2), with or without IPTG induction, or in theuninduced cultures of the putative specific clones. The presence andabsence of a peptide with identical molecular mass to authentic celeryM6PR (FIG. 2B), which also reacted with M6PR-specific antisera (FIG.2C), correlated well with the measured enzyme activities. Trace amountsof M6PR peptide were detected in uninduced cultures of the specificclones (FIG. 2C) but the levels were below that detectable in the enzymeassay (FIG. 2A). This experiment was repeated twice with identicalresults.

Sequencing M6PR Clones

EcoR1-Kpn1 restriction digestion of the three putative M6PR clonesyielded inserts of approximately 1.3 kb. The sequence of one of theseinserts is shown in FIGS. 3A and 3B. The sequence of the two otherputative clones was also determined (data not show). Each of thesequenced inserts coded for a 927 bp open reading frame; the clonesdiffered from each other in the length of the 5′ non-coding region andthe poly-A tail, suggesting that they represent independent cloningevents rather than duplications arising during library amplification.Translation of the open reading frame yielded a polypeptide with amolecular mass of 35.2 kD (FIGS. 3A and 3B). This value was consistentwith the previously determined value for authentic celery M6PR(determined by SDS-PAGE) of 34.5 kD (Loescher et al., Plant Physiol.98:1396-2401 (1992)) and with MALDITOFMS determined values for therecombinant and authentic M6PR protein (see below, Table 3). Thepredicted translation product was also consistent with an internalpeptide sequence obtained after tryptic digestion of purified celeryM6PR. The predicted translation product was identical to the internalamino acid sequence R, S, I, L, D, D, E, G (Arg Ser Ile Leu Asp Asp GluGly) (FIGS. 3A and 3B). A search of the non-redundant data base at NCBIgave only one entry showing homology (Mycoplasma pneumoniae M129B18cytadherence-accessory) indicating the rarity of this specific sequence.

Sequence Comparisons

Sequence similarities resulting from a search of the NCBI non-redundantdata base resulted in 61 entries showing greater than 55% sequencehomology with either the whole length of defined regions of the M6PRORF. This information and an analysis of the amino acid sequence of M6PRthrough two motif analysis programs (see Materials and Methods) showedM6PR to be a member of the aldoketo reductase family. A detaileddescription of the features and members of this group can be gained fromBohren et al., J. Biol. Chem. 264:9547-9551 (1989). In brief, the groupis typified by the three conserved domains marked in FIGS. 3A and 3B.

Five sequences of plant origin were obtained from the nucleotidesequence comparison (listed in Table 1)

TABLE 1 Sequence similarity between M6PR and entries accessible throughthe National Institute of Biotechnological Information (NCBI) data base.Accession % # Clone Identity Comparable sequence Identity D11080 AppleS6PDH Full mRNA 66 NADP-dependent mRNA Z48383 Arabidopsis thaliana bases14-325 70 315 bp EST of M6PR ORF D41273 Oryza sativa (Rice) bases 3-29167 462 bp EST of M6PR ORF D48175 Oryza sativa bases 118-331 73 430 bpEST of M6PR ORF X57526 Hordeum vulgare (Barley) Bases 413-635 53 aldosereductase

A direct sequence comparison with NADP-dependent sorbitol-6-phosphatedehydrogenase, which showed the greatest degree of homology over theentire M6PR sequence, is given in FIGS. 4A and 4B.

Purification and Characterization of Recombinant M6PR

Table 2 shows the results of a typical purification of M6PR from cloneD.

TABLE 2 Summary of a typical purification of recombinant M6PR from a 1liter, IPTG induced, culture of clone D. Total Specific Purifi- volprotein Activity Activity Yield cation STEP (ml) (mg) (mU) (mU/mg prot)(%) (X) Crude 22.5 178 13433  75 100 1 30-60% 10 79 14155 179 105 2.4acetone S200 gel filt. 67 77 12663 164  94 2.18 RY 86, 4 0.5  2216 4432  16 59 2 passes 1 mU = 1 nmol NADPH oxidized per minute. RY 86 =Reactive Yellow 86 affinity chromatography. On the first pass M6PR waseluted with 0.1 mM NADPH, on the second pass M6PR was eluted with alinear gradient of 0 to 0.2 mM NADPH. At the end of the purification theenzyme was concentrated and NADPH was removed by ultrafiltration.Buffers used during the purification and the assay conditions were asdescribed in Loescher et al., 1992.

With RY86 purification and desalting, a 50 to 60 fold purification wasachieved. The average specific activity of the purified recombinantenzyme, 3926±833 mU/mg protein (average of three preparations; final twopreparations 4441±13 mU/mg), was comparable to that of purified celeryM6PR (3756 mU/mg protein, Loescher et al., Plant Physiol. 98:1396-1402(1992)). FIG. 5A shows an SDS-PAGE of the various purification steps.After the second pass over the RY86 column the dominant peptide, whichhad a molecular mass identical to authentic celery M6PR (FIG. 5A) andcross reacted with M6PR-specific antisera (FIG. 5B) represented 88±4% ofthe protein present (estimated by scanning densitometry; mean of 2independent preparations).

Characterization of Recombinant M6PR

Table 3 shows some characteristics of M6PR purified from the twosources.

TABLE 3 A comparison of the characteristics of purified leaf andrecombinant M6PR. Authentic Recombinant leaf M6PR M6PR SDSPAGEdetermined 34.8 ± 0.4 (2;3) 34.3 ± 0.4 (2;3) Molecular mass. (kD)MALDITOFMS determined 35.21 (1;1) 35.3 ± 0.01 (1;3) Molecular mass. (kD)V_(max); mannose 6-P 6.8 ± 1.3 (2;2) 8.0 ± 1.9 (4;6) (μmol mg prot⁻¹min⁻¹) k_(m); mannose 6-P 13.6 ± 2.7 (2;2) 10.1 ± 1.4 (4;6) (mM)V_(max); NADPH 3.7 ± 0.7 (2;2) 6.0 ± 2.3 (2;3) (μmol mg prot⁻¹ min⁻¹)k_(m); NADPH 2.1 ± 1.1 (2;2) 6.2 ± 2.4 (2;3) (μM) SDSPAGE determinedmolecular masses of purified authentic and recombinant M6PR weredetermined by running them in adjacent wells on 12.5% acrylamide gels.MALDITOFMS = Matrix Assisted Laser Desorption Ionization Time Of FlightMass Spectrometry; the matrix was sinapinic acid. The values withinparentheses represent the number of individual enzyme preparations andthe number of independent determinations used to calculate the mean ofeach parameter, respectively. Kinetic parameters were determined at 30°C. in 33 mM Tris-HCl buffer (pH 7.5) containing 3 mM DTT (see Loescheret al. (1992) for other details concerning the assays). Mannose6-phosphate (M6P) kinetics were determined at 12 concentrations of M6Pranging from 1-50 mM under saturating NADPH concentrations (200 μM).NADPH kinetics were determined at 9 NADPH concentrations ranging from1-50 μM. Mannose 6-phosphate concentrations in these assays were 12 mM.Best line fits on double reciprocal plots were calculated by linearregression. At least 4 data points were used for the regression analyses(in most cases more than 8); r² values were > 0.99 except for onerecombinant NADPH determination

The molecular masses of the authentic and the recombinant proteinsdetermined by either SDS-PAGE or MALDITOF were almost identical and bothwere consistent with the value of 35.2 kD determined by translation ofthe coding region (see above). The V_(max) and k_(m) values determinedfor mannose 6-phosphate were the same for both enzymes (Table 3) andthose determined for NADPH were not significantly different (by t-test).

In this Example 1 shows the successful cloning of a full lengthtranscript coding for mannose 6-phosphate reductase from celery. Thisrepresents the first report of the cloning of the purification ofcompetent recombinant enzyme from transformed E. coli. The purifiedrecombinant protein had physical and kinetic propertiesindistinguishable from the purified plant enzyme.

The authenticity of the clones was confirmed by the following criteria.1). Only putative clones displayed M6PR activity when induced with IPTG.The activity correlated with the presence of a peptide, of identicalmolecular mass to authentic celery leaf M6PR (Table 3). This peptidecross reacted with M6PR-specific antisera (FIG. 2C). No activity orimmuno reactive peptide was observed in non-specific clones. 2). Atryptic digestion product from authentic celery M6PR had 100% homologyto a peptide present within the open reading frame of the putative clone(FIGS. 3A and 3B). 3). Database comparisons showed that the clones had ahigh degree of homology to the aldo-keto reductase family. The greatestdegree of homology (spanning the whole ORF with 67% sequence and 64%amino acid identity; 84% similarity when amino acids with the samefunctional properties were considered (FIGS. 4A and 4B)) was withNADP-dependent D-sorbitol 6-phosphate dehydrogenase (Kanayama et al.,Plant Physiology 10:1607-1608 (1992)), a key enzyme in sorbitolbiosynthesis in woody Rosaceae species. The sequence and amino acidsimilarity was very low (<22%) on an amino acid and sequence comparisonbasis) between M6PR and mannitol dehydrogenase (MTD; Williamson et al.,Proc Natl. Acad. Sci. 92:7148-7152 (1995)), the only other mannitolmetabolizing enzyme cloned to date from higher plants. However, fourother similar plant sequences were obtained from the databases. Three ofthese were for partial sequences from arabidopsis and rice which showedstrong homology with the 5′ end of M6PR (Table 1). Although there arenot known to be any reports of sugar alcohols in arabidopsis and rice,the recent discovery of a homologue of mannitol dehydrogenase inarabidopsis (Williamson et al., Proc. Natl. Acad. Sci. 92:7148-7152(1995)) may indirectly confirm Bieleski's admonition that the presenceof sugar alcohols should not be discounted until proven absent(Bieleski, R. L., Sugar Alcohols. In: F. A. Loewus and W. Tanner, eds.,Plant carbohydrates I. Intracellular Carbohydrates, Encyc. PlantPhysiol., Vol. 13A, New Series. Springer-Verlag, NY, pp. 158-192(1982)). The fifth plant enzyme sequence with similarity to M6PR was analdose reductase from barley (see below). As mentioned, there is almostno information as to how sugar alcohol metabolism is regulated andintegrated with the other products of primary production (sucrose,starch and nitrogen metabolism) in higher plants. In contrast, there isa relatively large body of work on the kinetics and regulation of animalaldose reductase, driven by their putative role in the pathology ofdiabetes mellitus (Borhani, D. W., et al., J. Biol. Chem.267:24841-24847 (1992)). The sequence homologies suggest that insightsinto regulatory sites and mechanisms in the plant enzymes may be gainedfrom the animal literature. For example, several sites are highlyconserved between the animal and plant enzymes (marked as motifs 1, 2and 3 in FIG. 3B) including the peptide IPKS (within motif 3, towardsthe 3′ end of the coding region). The lysine (K) within this motif hasbeen shown, by chemical modification studies in animals, to be thelikely NADPH binding site (Morijana et al., FASEB J 46:1330 (abstract)(1987); Bohren et al., J. Biol. Chem. 264:9547-9551 (1989)). This motifis completely conserved in terms of amino acids and position in M6PR(IPKS, amino acids 260-264), NADP-dependent D-sorbitol 6-phosphatedehydrogenase (IPKS, amino acids 260-264) and in aldose reductase (IPKS,amino acids 257-260) cloned from desiccated barley embryo's (Bartels etal., EMBO J 10:1037-1043 (1991)). This latter aldose reductase has beenassociated with tissues subjected to desiccation and is inducible withABA which is of interest given the role that mannitol synthesis and M6PRplay in salinity stressed celery (Everard et al., Plant Physiol.106:281-292 (1994); Loescher et al., Plant Physiology 170-178 (1995)).Another similarity between the animal aldose reductases and M6PR is theapparent lack of post-translational modification. In vitro translationof celery poly(A)+RNA resulted in a peptide immunoprecipitation productof identical molecular mass to authentic leaf M6PR (FIG. 1B) indicatingthat post-translational modification is unlikely to occur in vivo, aconclusion also drawn for bovine lens aldose reductase (the model forstudy of this class of enzymes prior to the availability of recombinantenzymes, Schade et al., J.B.C. 265(7):3628-3635 (1990). Finally, aldosereductases in animals are sensitive to oxidation (Petrash et al., JBC267(34):24833-24840 (1992)), as is the M6PR (Loescher et al., PlantPhysiol. 98:1396-1402 (1992)), and the current thinking is that redoxactivation/inactivation may play an important role in the in vivoregulation of M6PR. The importance of redox activation of extraplastidicplant enzymes has grown in recent years and an increasing number ofcytosolic (Anderson et al., Planta 196:118-124 (1995)) and mitochondrialenzymes are being reported to be regulated in part by this mechanism.The recombinant enzyme has a specific activity similar to the plantenzyme which suggests that if redox activation is a factor then the E.coli thioredoxin system is competent. Such information should lead toapproaches to look for regulatory mechanisms in the plant enzymes,studies that will be simpler with the recombinant enzyme which appearsfully competent and kinetically indistinguishable from the authenticenzyme. Ultimately, mechanisms may be explored at the enzyme level usingsite directed mutagenesis and at the whole plant level by studies intomessage level regulation, such as that recently reported forNADP-dependent D-sorbitol 6-phosphate dehydrogenase (Kanayama et al.,Plant Physiology 100:1607-1608 (1995)), and by pathway suppression usingantisense and cosuppression techniques. Such studies should give insightinto the roles of polyols in primary carbon metabolism and hence plantproductivity as well as in stress tolerance.

DNA is incorporated into plants in a manner known to those skilled inthe art as represented by U.S. Pat. No. 5,492,820 to Sonnewald et al.Various well known vectors are used.

EXAMPLE 2

A large number of techniques are available for inserting M6PR DNA into aplant host cell. Those techniques often include transformation withT-DNA using Agrobacterium tumefaciens or A. rhizogenes containing the Tior Ri plasmids (respectively) as transformation agents. Some of theother methods used include fusion, biolistic or conventional injection,or electroporation. If Agrobacterium related methods are used, the DNAis cloned into a special plasmid, either an intermediate vector or intoa binary vector. Intermediate vectors are integrated into the Ti or Riplasmid by homologous recombination resulting from sequences that arehomologous to sequences in the T-DNA. The Ti or Ri plasmid alsocomprises the vir region necessary for transfer of the T-DNA. Theintermediate vector are transferred into Agrobacterium by means of ahelper plasmid (via conjugation). Binary vectors replicate themselvesboth in E. coli and Agrobacterium. These vectors include a selectionmarker gene and a linker or polylinker that are framed by the right andleft T-DNA border regions. These are transformed directly intoAgrobacterium and the Agrobacterium is then used as a host cell for theplasmid carrying the vir region. The vir region is also necessary forthe transfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The transformed bacteria are used for transformation of plantcells. Plant explants (e.g., sections of leaves, stems and roots,segments of petioles, flowers, and flower parts) are cultivated withAgrobacterium tumefaciens or A. rhizogenes for the transfer of the DNAinto the plant cell. Whole plants are then regenerated (from theinfected plant material, or from protoplasts or suspension-cultivatedcells) in a suitable medium which can contain antibiotics or biocides(e.g., kanamycin, bleomycin, hygromycin, chloramphenicol, among others)for selection of transformed plant cells. Unlike Agrobacterium-mediatedinsertion of M6PR DNA, no special demands are necessary for constructionfor plasmids used for particle bombardment, fusion, injection, orelectroporation. It is possible to use ordinary plasmids, e.g., pUCderivatives, although selection markers are usually included. Howeverobtained, whole plants are then tested for the presence of the insertedDNA. The transformed cells grow normally in the plant and eventuallygive rise to reproductive organs, i.e., flowers, that can be used in anordinary breeding program. The resulting hybrids have the appropriatephenotypic properties.

Expression of the M6PR DNA requires a promoter associated with thecloned gene. Among the many examples available are viral promoters suchas the cauliflower mosaic virus 35 S promoter, heat shock proteinpromoters such as the HSP 70 promoter, light induced promoters such asthe ST-Ls1 or the rubisco small subunit (SSU) promoters, stress responseproteins such as the PR protein promoter, the Agrobacterium tumefaciensnos promoter, and various organ, root, tuber (e.g., the class Ipatatin), and leaf specific promoters. A termination signal is also usedin these constructs, e.g., the 31′-end of the poly-A side of theoctopine synthase gene.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

We claim:
 1. An isolated and purified DNA encoding mannose-6-phosphatereductase (M6PR) as set forth in SEQ ID NO:1.
 2. An isolated andpurified DNA encoding mannose-6-phosphate reductase (M6PR) in a plasmidin an Escherichia coli as contained in the deposit identified as ATCC98041.
 3. A recombinant plasmid containing DNA encodingmannose-6-phosphate reductase as set forth in SEQ ID NO:1.
 4. Arecombinant plasmid containing DNA encoding mannose-6-phosphatereductase in a plasmid in E. coli as contained in the deposit identifiedas ATCC
 98041. 5. A bacterium containing a recombinant plasmidcontaining DNA encoding mannose-6-phosphate reductase as set forth inSEQ ID NO:1.
 6. A bacterium containing a recombinant plasmid containingDNA encoding mannose 6-phosphate reductase in the plasmid in an E. colias contained in the deposit identified as ATCC
 98041. 7. A method forproducing mannose 6-phosphate reductase (M6PR) which comprises: (a)providing a bacterium containing a recombinant DNA encodingmannose-6-phosphate reductase (M6PR) as set forth in SEQ ID NO:1 in aculture medium; (b) expressing the M6PR in the culture medium; and (c)isolating the M6PR.
 8. A method for producing mannose 6-phosphatereductase (M6PR) which comprises: (a) providing a bacterium containing arecombinant plasmid containing DNA encoding mannose 6-phosphatereductase in the plasmid in an E. coli as contained in the depositidentified as ATCC 98041 in a culture medium; (b) expressing the M6PR inthe culture medium; and (c) isolating the M6PR.